The analysis of thermal non-uniformity (TNU) is an established attribute of the art for characterizing the performance of a thermal block assembly, which may be used in various bio-analysis instrumentation. TNU is typically measured in a sample block portion of a thermal block assembly, which sample block may engage a sample support device. TNU may be expressed as either the difference or the average difference between the hottest and the coolest locations in the sample block. For example, TNU may be determined as a difference or average difference between a hottest and a coldest sample temperature or position in a sample block. An industry standard, set in comparison with gel data, may express TNU so defined as a difference of about 1.0° C., or an average difference of 0.5° C. Historically, the focus on reducing TNU has been directed towards the sample block. For example, it has been observed that the edges of the sample block are typically cooler than the center, and this difference in temperature is transferred to a biological sample being processed in the sample support device.
One of the common reasons for non-uniformity across a plurality of samples, particularly when placed in an array of wells, is referred to in the art as edge effects. Edge effects typically occur in configurations where the wells at the outer edges of a microtiter plate, for example, release heat to the ambient more rapidly than the wells located in the center of the microtiter plate. This results in a temperature differential between the center wells and the outer wells. This effect is exacerbated by water in the biological sample evaporating inside the well and condensing on the inner wall of the well above the biological sample. One skilled in the art would realize that a loss of fluid in the biological sample alters the concentration of the reactants in the biological sample and also affects the pH of the reaction. Both the change in concentration and pH affect the efficiency of the reaction resulting in non-uniform reaction efficiencies across the wells of the microtiter plate and therefore, the biological samples.
Various embodiments of a sample block may be adapted to receive various sample containing devices, such as a microtiter plate. Additionally, various embodiments of a sample block may have a substantially flat surface adapted to receive a substantially planar sample-containing device, such as a microcard. In a sample block capable of receiving a microtiter plate or microcard or any other vessel suitable for nucleotide processing, biological samples deposited in the vessels may undergo thermal cycling according to a thermal cycling profile. For example, a two setpoint thermal cycling profile may include a setpoint temperature for a denaturation step and a setpoint temperature for an annealing/extension step. Setpoint temperatures for a denaturation step may be between about 94-98° C., while setpoint temperatures for an annealing/extension step may be between about 50-65° C. Alternatively, three setpoint temperature protocols can be used, in which the annealing and extension steps are separate steps. According to various protocols, the setpoint temperature for an extension step may be between about 75-80° C. During the defined steps of a thermal cycle, in order to allow time for the chemical process at that step, a specified hold time for the setpoint temperature may be defined. One of ordinary skill in the art is apprised the hold times for various steps in a thermal cycle may be for different intervals. For all protocols, regardless of the setpoint temperature protocol used, one of ordinary skill in the art would understand that the success or failure of the protocol depends, at least in part, on a thermal cycler achieving the desired temperature of each setpoint, and each well containing a biological sample being subjected to that setpoint temperature throughout the hold time as mentioned above.
It is important for one of ordinary skill in the art to be able to determine the thermal non-uniformity of a sample block assembly. A common approach is to use, for example, thermocouples, thermistors, PRTs or other types of thermal sensors well known in the art. The sensors are used to detect temperatures at various points across an array of sample vessels. The measured temperatures are then used to calculate temperature non-uniformity and compare the result to the accepted values as discussed above.
In the present teachings, the effects of condensation and evaporation of aqueous components of the biological samples, were discovered to be a significant factor contributing to temperature non-uniformity of thermal block assemblies currently available and in use within the bio-analysis research community. The present teachings present an innovative approach to controlling the condensation and evaporation of the aqueous components in biological samples, which embodiments according to the present teachings are in contrast to various established teachings of the art.